Triterpenes and Steroids from Euphorbia denticulata Lam. With Anti-Herpes Symplex Virus Activity.

In this research, dried acetone:chloroform extract of aerial parts of E. denticulata as one of the endemic plants to Iran, afforded a number of triterpenes and steroids including: betulin, 24-methylene-cycloart-3-ol, cycloart-23Z-ene-3β,25-diol, cycloart-23E-ene-3β,25- diol, ergosta-8,24-dien-3-ol (obtusifoliol) and beta-sitosterol which were reported for the first time from this plant. The structure of these compounds was elucidated by NMR and mass spectroscopic methods. The MTS assay was used to determine the toxicity and antiviral activity of betulin and (3β,23E)-cycloarta-23-ene-3,25-diol. Betulin showed anti-HSV-1 activity with EC50 value of 84.37±0.02 μg/mL, and toxicity on normal vero cells with CC50 value of 660.718±0.072 μg/mL. (3β,23E)-Cycloarta-23-ene-3,25-diol showed antiviral effect with EC50 value of 86.63±0.03 μg/mL, and toxicity with CC50 value of 1089.21±0.25 μg/mL. The results revealed that these two compounds have the antiviral activity far below the CC50 value with selectivity index (CC50/EC50) values of 7.83, and 12.57, respectively.


Introduction
Euphorbiaceae is one of the largest families of the phylum Anthophyta. In this family the largest genus is Euphorbia which comprises well over 2000 species in tropical and temperate zones of Asia and other parts of the world (1). In Iran 70 species are reported that 17 of them are endemic. In traditional medicine Euphorbia was used to treat inflammations or as an antivirus or antitumor (2, 3). There are reports on Euphorbia species with anti HIV effects or anti herpes simplex virus (3)(4)(5). Currently coordinate with the medicinal chemists which rapidly build and develop new synthetic drugs, researchers of the natural product chemistry are also discovering secondary metabolites in plants with their subsequent biological effects. In this context, the paper in hand was also aimed to isolate and Thin layer chromatography detection was achieved by spraying the silica gel plates with cerium sulfate in 10% aq. H 2 SO 4 , followed by heating.

General experimental procedures
The NMR spectra were recorded on a Bruker Avance AV 400, using CDCl 3 as solvent. HPLC was carried out on a waters 515 using a Pack-Sil column (250×20 mm i.d.) packed with 5 µm silica (YMC Co., Ltd., Kyoto, Japan) and hexane:EtOAc as mobile phase. Chromatographic materials were silica gel (Merck Co., Germany). cell repository of tissue culture department, Pasture Institue, Iran. Herpes simplex virus type 1 (HSV-1, strain KOS) obtained from Virology Department of Tarbiat Modares University (Tehran, Iran).

Plant material
Plant material was collected from populations growing in Sanandaj (Iran) at the West part of Iran and identified by Dr. Hojatollah Saeedi in the Department of Biology, Faculty of Science, University of Isfahan and a voucher specimen (#19001) is preserved in the herbarium of the University of Isfahan (Iran).

Virus and cells
For cytotoxicity and antiviral assays cells were grown in Dulbeccoʼs Modified Eagleʼs growth Medium ( DMEM ; Sigma, USA) supplemented with 2% of fetal bovine serum (FBS; Gibco, Germany), 100 µg/mL of streptomycin, 100 UI/ mL of penicillin and 0.25 µg/mL amphotericin B ( Gibco, Germany) and 0.14% (v/v) sodium bicarbonate. All the cells were cultured at 37°C in a humidified atmosphere supplied with 5% CO2.
Stock preparations of the herpes simplex virus type 1 (HSV-1, strain KOS), were generated by incubating in Vero cells (75 cm 2 culture flasks seeded with 350000 cells/mL). After 72 h infection, the cultures were frozen and thawed twice before centrifugation and the resulting supernatant aliquots stored at -70°C. Virus titers were determined by cytopathic effects in Vero cells and were expressed as 50% Tissue Culture Infective Dose (TCID50) per mL (7, 8).

Cytotoxicity assay
The Vero cells were seeded onto a 96well plate at a concentration of 3.5×10 5 Vero cells per mL and a volume of 100 µL per well. Following 24 h incubation at 37 °C, a confluent cell monolayer was confirmed and cell media was removed. Test compounds were serially diluted with the culture medium supplemented with 2% serum to reach the different concentrations. Negative control dilution of DMSO at 0.1 % was also included. An aliquot of 100 μL/well of each diluted compound or DMSO was added to the plates in triplicate. After incubation at 37 °C with 5% CO 2 for 3 days, MTS (Cell Titer 96; Promega, USA) was added to each well with a volume of 20 µL. The trays were further incubated for 2 h to allow MTS production. The absorbances were determined with an ELISA reader (Stata Fax 2100, USA) at a test wavelength of 490 nm. Data were calculated as the percentage of inhibition using the following formula: inhibition % =[100 -( A t / A s ) × 100]%. A t and A s refer to the absorbances of the test substances and the solvent control, respectively. CC50 values, defined as the concentration of 50% cellular cytotoxicity (CC50) of test compounds (7, 8).

Antiviral assay using MTS method
The antiviral activity of compounds isolated from E. denticulata against HSV-1 were evaluated by the MTS method. Vero cells, treated with trypsin, were seeded onto 96-well plates with a concentration of 5 × 10 3 cells in a volume of 100 µL per well. After incubation at 37 °C with 5% CO2 for 24 h, when the cell monolayers were confluent, the medium was removed from the wells and 100 µL of test virus was added and incubated for another 2 h. Different noncytotoxic concentrations ( ≤ CC50 values) of test compounds were then added to culture wells in triplicate. The maximum concentration of DMSO (0.1%) was used as a negative control. Aciclovir; Sigma, USA) was used as a positive control for HSV-1. After incubation at 37°C with 5% CO2 for 3 days, the MTS test was carried out as previously described. The percentages of protection were calculated as [(A − B) × 100/( C−B)], where A, B, and C indicate the absorbances of test compounds, virus and cell controls, respectively. Each obtained EC50 value was defined as the effective concentration that reduced the absorbance of infected cells to 50% when compared with cell and virus controls (7, 9).

Time-course anti-virus antanalysis of isolated compounds
Different non-cytotoxic concentrations (≤ CC50 values) of test compounds were added to culture cells in triplicate at different times pre-infection or post-infection. HSV-1(10 TCID50 per well) was inoculated onto confluent monolayers of Vero cells for 2 h. After 3 days, MTS test and antiviral activity were carried out as previously described (3).

Statistical analysis
The selectivity index (SI) was determined as the ratio of CC50 to EC50.The statistically different effects of test compounds on the inhibition of HSV -1 were compared with the control group or together using the Student 's t-test.  structure of compound 1 detected as betulin (

Assessment of cytotoxicity and anti-HSV-1 activity by MTS assay on Vero cell
The MTS assay was used to determine the toxicity and antiviral activity of the tested agents (9, 16). Betulin and (3 β,23E)-Cycloarta-23ene-3,25-diol isolated from E.denticulata were investigated for anti-HSV-1 activity, were firstly tested for their cytotoxic effect alone on Vero cells. The results revealed that both compounds have antiviral activity far below the CC50 doses (Table 2). Results presented in Table 2 revealed that the CC50 of betulin and (3β,23E)-Cycloarta-23-ene-3,25-diol were 660.718±0.072 and 1089.205±0.250 µg/mL, respectively. The results also showed that the rate of cells death increased with increasing the concentration of the tested compounds. After studying the cytotoxicity effect of samples on normal Vero cells, the experiments to assess the antiviral effect of samples on HSV-1 were done.

Time-course anti-virus analysis of isolated compounds
In order to investigate the mechanism of how each compound inhibits the infection of HSV-1 a study was conducted to investigate the time-course effect at 1 h before to 24 h after the virus infection and using different concentrations of each compound (1, 10, 100 µg/mL). Inhibition was evaluated by MTS assay after 3 days of infection and expressed as percentage inhibition. As it is clear from Figure 3a and Figure 3b, the both compounds betulin and (3β,23E)-Cycloarta-23-ene-3,25diol, exhibited the highest inhibition against HSV-1 infection within 2.0 h post-infection which were during the early period of virus replication.

Conclusion
E. denticulata as one of the endemic plants to Iran, could be a new source of 4,4 dimethyl steroids like 24-methylene-cycloart-3-ol, cycloart-23Z-ene-3β,25-diol, and cycloart-23E-ene-3β,25-diol as well as obtusifoliol as 4α-methyl steroid and beta-sitosterol as 4-desmethyl steroid which were reported for the first time from this plant. In addition E. denticulata could be considered as one on the economic sources of betulin (0.01 % of dry weight of the plant).